Summary of Work: Drosophila melanogaster maintains telomere length by a process of tip specific transposition. Two non-LTR retrotransposons, HeT-A and TART, are known to be responsible for telomere elongation in this organism. Based on in situ hybridizations, HeT-A is expressed in actively dividing cells, especially in the germ line. In standard laboratory strains only about 1% of chromosome ends are extended per sexual generation, suggesting that transcription is not the limiting step in transposition. A few strains have terminal HeT-A arrays much longer than normal. A genetic factor on chromosome 3 has been identified that induces terminal HeT-A arrays to grow. Unlike other retroposons, HeT-A doesn?t encode its own reverse transcriptase (RT), a key enzyme in retrotransposition. To investigate the source of the HeT-A RT, we have adapted a biochemical assay to measure RT activity in cell extracts and in situ. Preliminary data suggest that the RT activity is higher in the strains with longer telomeres. To investigate the source of RT genetically and to show that this element transposes via an RNA intermediate as expected from its sequence, we have prepared plasmid constructs with HeT-A sequence and two reporter genes. The white-eye gene, interrupted with a reversed intron, will be expressed only after HeT-A transposition. Embryo injections to produce transformants are in progress. Once we can quantify transposition, these elements will be used to characterize the genetic factors that stimulate HeT-A transposition. A gene for Green Fluorescent Protein (GFP) will also be used as a reporter for transposition to relate cells expressing HeT-A mRNA to those that allow transposition. Similar constructs are being made using L1, a human non-LTR retrotransposon. A number of stably transformed cell lines are being used. Two of these cell lines are human colon cancer cell lines that are mutant for mismatch repair genes, hMSH6 and hMLH1. Experiments are underway to measure transposition frequency in the mutant cell lines. Another cell line mutant for p53 is under investigation.